de Almeida João Ricardo Moreira, de Moraes Lídia Maria Pepe, Torres Fernando Araripe Gonçalves
Laboratório de Biologia Molecular, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF 70910-900, Brazil.
Yeast. 2005 Jul 15;22(9):725-37. doi: 10.1002/yea.1243.
We report the cloning of the 3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris by a PCR approach. The coding sequence of the PGK1 gene comprises 1251 bp with the potential to encode a polypeptide of 416 amino acid residues, which shows high identity to homologous proteins from other yeasts. The promoter region of this gene (P(PGK1)) contains regulatory cis-elements found in other PGK1 genes, such as TATA box, CT-rich block and a heat shock element. In the 3' downstream region we identified a tripartite element 5'-TAG-TAGT-TTT-3', which is supposed to be important for transcription termination. As in other yeasts, the PGK1 gene from P. pastoris is present as a single-copy gene. Northern blot analysis revealed that the gene is transcribed as a 1.5 kb mRNA; when cells are grown on glucose the levels of this mRNA are increased two-fold in comparison to cells grown on glycerol. The transcriptional regulation of this gene by the carbon source was further confirmed when the alpha-amylase gene from Bacillus subtilis was placed under the control of P(PGK1): higher levels of expression were obtained when cells were grown on glucose as compared to glycerol and methanol. Preliminary results related to the strength of P(PGK1) show that it represents a potential alternative to constitutive heterologous expression in P. pastoris.
我们报道了通过PCR方法从甲基营养型酵母毕赤酵母中克隆3-磷酸甘油酸激酶基因（PGK1）。PGK1基因的编码序列包含1251个碱基对，有可能编码一个由416个氨基酸残基组成的多肽，该多肽与其他酵母的同源蛋白具有高度同源性。该基因的启动子区域（P(PGK1)）包含在其他PGK1基因中发现的调控顺式元件，如TATA盒、富含CT的区域和一个热休克元件。在3'下游区域，我们鉴定出一个三联体元件5'-TAG-TAGT-TTT-3'，据推测它对转录终止很重要。与其他酵母一样，毕赤酵母的PGK1基因以单拷贝基因形式存在。Northern印迹分析表明该基因转录为1.5 kb的mRNA；当细胞在葡萄糖上生长时，这种mRNA的水平比在甘油上生长的细胞增加了两倍。当将枯草芽孢杆菌的α-淀粉酶基因置于P(PGK1)的控制下时，进一步证实了该基因受碳源的转录调控：与在甘油和甲醇上生长相比，当细胞在葡萄糖上生长时获得了更高水平的表达。与P(PGK1)强度相关的初步结果表明，它是毕赤酵母中组成型异源表达的一种潜在替代方案。
